Method of modulating microglial activation for the treatment of acute and chronic neurodegenerative disorders

ABSTRACT

The present invention provides methods of modulating or inhibiting microglia activation comprising the administration of a compound capable of inhibiting 5-LOX, FLAP, attenuating degradation of IKBα or inhibiting nuclear translocation of the NF-KB active complex for the treatment of Alzheimer&#39;s disease, brain ischemia, traumatic brain injury, Parkinson&#39;s Disease, Multiple Sclerosis, ALS, subarachnoid hemorrhage or other disorders associated with excessive production of inflammatory mediators in the brain.

CROSS REFERENCE TO RELATED APPLLICATIONS

[0001] This application is a divisional application of nonprovisionalapplication U.S. Ser. No. 09/899,657 filed Jul. 5, 2001 which claimspriority from provisional applications U.S. Ser. No. 60/218,085 filedJul. 13, 2000 and USSN 60/261,332 filed Jan. 12, 2001.

FIELD OF THE INVENTION

[0002] The present invention comprises methods of treating various acuteand chronic central nervous system disorders by the administration ofFLAP or 5-lipoxygenase inhibitors.

BACKGROUND OF THE INVENTION

[0003] Acute and chronic brain injuries can activate resident microglia(resident macrophage-like cells found in the central nervous system) aswell as recruit peripheral immune cells to injured brain regions thatcan exacerbate neuronal damage. Inflammatory processes can induce celldeath by (a) the release of proteases and free radicals that inducelipid peroxidation, (b) direct cytotoxic effects or (c) by thephagocytosis of sublethally injured neurons. The attenuation ofmicroglia and peripheral immune cell activation has been correlated withsignificant neuronal protection in pre-clinical studies of ischemia,traumatic brain injury, spinal cord injury and Alzheimer's disease.

[0004] Oxygenase enzymes like cycloxygenase and lipoxygenase caninitiate the conversion of arachidonic acid to physiological importantmetabolites. Cycloxygenase (COX; prostaglandin H2 synthase) isresponsible for the formation of prostaglandins and thomboxanes. SeeVersteeg, H. Van, van Bergen en Henegouwen, M. P. V., van Deventer, S.J. W. and Peppelenbosch, M. P. (1999). Cyclooxygenase-dependentsignaling: molecular events and consequences. FEBS letters 445: 1-5.Lipoxygenase is responsible for the conversion of arachidonic acid toleukotrienes. Lipoxygenases and Their Metabolites, Plenum Press, NewYork. Eds. Nigam and Pace-Asciak. (1999). It is hypothesized thatprostaglandins are an important step in transducing immune stimuli intoCNS responses. There are two known isozymes of COX currently known COX-1(constituitively expressed) and COX-2 (induction in response to immunestimuli). It has been established that COX-1 and COX-2 are found to beinduced and constituitively expressed in peripheral immune cells as wellas brain, with neuronal expression of COX-2 being enhanced followingvarious CNS insults including cerebral ischemia. Tomimoto, H., Akiguchi,I. Watkita, H., Lin, J. X., Budka, H. Cyclooxygenase-2 is also inducedin microglia during chronic cerebral ischemia in humans. ActaNeuropathol (Berl) 1:26-30 (2000).

[0005] However, little is known about the role of lipoxygenases (orsubsequent metabolites including hydroxyeicosatetraenoic acids (HETEs),leukotrienes, lipoxines, and hepoxilins) in regulating braininflammation or neurodegeneration. There are currently four known humanlipoxygenases (5, 8, 12, and 15-lipoxygenase). All isoforms share acommon substrate as well as oxygenase activity but differ greatly insequence. Although, the role of prostaglandins and COX-2 in modulatinginflammation and pain has been well elucidated, the importance of LOXenzymes (specifically 5-LOX or 5-lipoxygenase) in brain following injuryis still unresolved. Simon, L. S. Role and regulation ofcyclooxygenase-2 during inflammation American Journal of Medicine106:37S-42S (1999).

SUMMARY OF THE INVENTION

[0006] Thus, according to a first embodiment of a first aspect of thepresent invention is provided a method of modulating or inhibitingmicroglia activation comprising the administration to a human in needthereof a compound capable of inhibiting 5-LOX.

[0007] According to another embodiment of the first aspect of thepresent invention is provided a method of modulating or inhibitingmicroglia activation comprising the administration to a human in needthereof a compound capable of selectively inhibiting 5-LOX over COX-2.

[0008] According to another embodiment of the first aspect of thepresent invention is provided a method of modulating or inhibitingmicroglia activation comprising the administration to a human in needthereof a compound capable of inhibiting FLAP.

[0009] According to another embodiment of the first aspect of thepresent invention is provided a method of modulating or inhibitingmicroglia activation comprising the administration to a human in needthereof para-REV5901 (L-655,238), Bay-x-1005, ML-3000, NDGA orZILEUTON®.

[0010] According to a first embodiment of a second aspect of the presentinvention is provided a method of inhibiting the release ofpro-inflammatory substances from activated microglial cells comprisingthe administration to a human in need thereof a compound capable ofinhibiting 5-LOX.

[0011] According to another embodiment of a second aspect of the presentinvention is provided a method of inhibiting the release ofpro-inflammatory substances from activated microglial cells comprisingthe administration to a human in need thereof a compound capable ofselectively inhibiting 5-LOX over COX-2.

[0012] According to another embodiment of a second aspect of the presentinvention is provided a method of inhibiting the release ofpro-inflammatory substances from activated microglial cells comprisingthe administration to a human in need thereof a compound capable ofinhibiting FLAP.

[0013] According to another embodiment of a second aspect of the presentinvention is provided a method of inhibiting the release ofpro-inflammatory substances from activated microglial cells comprisingthe administration to a human in need thereof para-REV5901 (L-655,238),Bay-x-1005, ML-3000, NDGA or ZILEUTON®.

[0014] According to a first embodiment of a third aspect of the presentinvention is provided a method of treating Alzheimer's disease, brainischemia, traumatic brain injury, Parkinson's Disease, MultipleSclerosis, ALS, subarachnoid hemorrhage or other disorders associatedwith excessive production of inflammatory mediators in the braincomprising the administration to a human in need thereof a compoundcapable of inhibiting 5-LOX.

[0015] According to another embodiment of a third aspect of the presentinvention is provided a method of treating Alzheimer's disease, brainischemia, traumatic brain injury, Parkinson's Disease, MultipleSclerosis, ALS, subarachnoid hemorrhage or other disorders associatedwith excessive production of inflammatory mediators in the braincomprising the administration to a human in need thereof a compoundcapable of 5-LOX over COX-2.

[0016] According to another embodiment of a third aspect of the presentinvention is provided a method of treating Alzheimer's disease, brainischemia, traumatic brain injury, Parkinson's Disease, MultipleSclerosis, ALS, subarachnoid hemorrhage or other disorders associatedwith excessive production of inflammatory mediators in the braincomprising the administration to a human in need thereof a compoundcapable of inhibiting FLAP.

[0017] According to another embodiment of a third aspect of the presentinvention is provided a method of treating Alzheimer's disease, brainischemia, traumatic brain injury, Parkinson's Disease, MultipleSclerosis, ALS, subarachnoid hemorrhage or other disorders associatedwith excessive production of inflammatory mediators in the braincomprising the administration to a human in need thereof para-REV5901(L-655,238), Bay-x-1005, ML-3000, NDGA or ZILEUTON®.

[0018] According to a first embodiment of a fourth aspect of the presentinvention is provided a method of attenuating degradation of IKBαcomprising the administration to a human in need thereof a compoundcapable of inhibiting 5-LOX.

[0019] According to another embodiment of a fourth aspect of the presentinvention is provided a method of attenuating degradation of IKBαcomprising the administration to a human in need thereof a compoundcapable of selectively inhibiting 5-LOX over COX-2.

[0020] According to another embodiment of a fourth aspect of the presentinvention is provided a method of attenuating degradation of IKBαcomprising the administration to a human in need thereof a compoundcapable of inhibiting FLAP.

[0021] According to another embodiment of a fourth aspect of the presentinvention is provided a method of attenuating degradation of IKBαcomprising the administration to a human in need thereof para-REV5901(L-655,238), Bay-x-1005, ML-3000, NDGA or ZILEUTON®.

[0022] According to a first embodiment of a fifth aspect of the presentinvention is provided a method of inhibiting nuclear translocation ofthe NF-KB active complex comprising the administration to a human inneed thereof a compound capable of inhibiting 5-LOX.

[0023] According to another embodiment of a fifth aspect of the presentinvention is provided a method of inhibiting nuclear translocation ofthe NF-KB active complex comprising the administration to a human inneed thereof a compound capable of selectively inhibiting 5-LOX overCOX-2.

[0024] According to another embodiment of a fifth aspect of the presentinvention is provided a method of inhibiting nuclear translocation ofthe NF-KB active complex comprising the administration to a human inneed thereof a compound capable of inhibiting FLAP.

[0025] According to another embodiment of a fifth aspect of the presentinvention is provided a method of inhibiting nuclear translocation ofthe NF-KB active complex comprising the administration to a human inneed thereof para-REV5901 (L-655,238), Bay-x-1005, ML-3000, NDGA orZILEUTON®.

[0026] Other embodiments of the invention comprise two or moreembodiments or elements thereof suitably combined.

[0027] Yet other embodiments and aspects of the invention will beapparent according to the description provided below.

DETAILED DESCRIPTION OF THE INVENTION

[0028] As used herein “a compound capable of selectively inhibiting5-LOX over COX-2” means a compound having 1 to 500-fold or more,particularly 1 to 50-fold and more particularly 1 to 10-fold selectivityfor 5-LOX over COX-2 as measured by the ability to attenuate theproduction of arachidonic acid metabolites from cellular suspensions(derived from blood or cell lines) stimulated with ionophore A23187 aspreviously described (Salari et al., 1984, Prostaglandins andLeukotrienes, Vol 13: 53-60; Menard et al., 1990, Br. J. Pharmacol 100:15-20) incorporated by reference herein. For instance, 5-HETE and LTB4are arachidonic acid metabolites derived from 5-LOX and12-hydroxy-heptadecatrienoic (HHT) is an arachidonic acid metabolite forcycloxygenase activity. Alternatively, COX-2 can be specificallyassessed by the ability to attenuate the production of the arachidonicacid metabolite, PGE2, from cellular suspensions (derived from blood orcell lines) stimulated with the LPS (Laufer et al., 1999, InflammationResearch, 48:133-138; Horton et al., 1999; Anal Biochim 271:18-28) .

[0029] As used herein “FLAP” means 5-LOX activating protein. Compoundsthat inhibit FLAP can be measured by the ability to inhibitphotoaffinity labeling of a source of purified FLAP (i.e. rat or human).In addition, FLAP inhibitors are confirmed if there is a correlation inthe inhibition of leukotriene synthesis in vitro cell based assays (i.e.Human PMN leukotriene synthesis) (Evans et al., 1991, MolecularPharmacology 40:22-27).

[0030] As used herein “inflammatory mediators in the brain” includes butis not limited to cytokines, chemokines, prostaglandins andleukotrienes.

[0031] As used herein “pro-inflammatory substances” includes but is notlimited to TNF-alpha, nitrite, NO, IL-6, IL-1, 5-HETE, LTB4, LTA4 andother inflammatory substances.

[0032] Bay-x-1005 (C₂₃H₂₃NO₃) is a selective inhibitor of FLAP. SeeDrugs Fut 1995, 20:996 and Drugs Fut 2000 25(10):1084.

[0033] ML-3000 is an inhibitor of both COX and LOX. See Drugs Fut 199520:1007 and Drugs Fut 25(10):1093.

[0034] REV5901-para-isomer (L-655,238- IC50=.luM- 5-LOX) is a selective5-lipoxygenase activating protein inhibitor (FLAP) with a quinolinestructure. It has been reported that FLAP inhibitors with this basicchemical structure interfere with 5-LOX and FLAP protein interactionspreventing a required cellular translocation of 5-LOX. Moreover, it hasbeen shown that compounds with the quinoline chemical structure do notaffect other routes of arachidonic acid metabolism including knowncycloxygenase and other lipoxygenases proteins (Evans et al., 1991,Molecular Pharmacology 40:22-27; Hutchinson, A.W. 1991, Trend inPharmacological Studies, 12: 68-70).

[0035] NDGA is a selective 5-lipoxygenese over cycloxgenase inhibitor(IC50=.2uM- 5-LOX, IC50=. 100 uM- COX)-Salari et al, 1984.

[0036] We have discovered that indirectly or directly inhibiting5-lipoxygenase can preferentially attenuate pro-inflammatory cytokinerelease from activated rat microglia cells in comparison to COX-2inhibition. While not intending to limit the scope of the invention toany particular mechanism the following description is provided.Cytosolic Ca2+dependent type IV phospholipase A2 (CPLA2) generatesintracellular arachidonic acid (AA). AA is converted to pro-inflammatoryprostaglandins, thromboxanes, and leukotrienes by either cycloxygenases(COX) or lipoxygenases (LOX).

[0037] Since cytosolic phospholipase A2 (cPLA₂) is one of the majorenzymes involved in the generation of AA, the effect oflipopolysaccharide (LPS) on cPLA₂ was determined. Indirectimmunofluorescence with a cPLA₂ specific monoclonal antibody revealedthat cPLA₂ was localized primarily in the cytosol in untreated cells.Upon stimulation with LPS, cPLA₂ redistributed to form punctate bodieswithin 15 minutes and returned to a control immuno-staining pattern by60 minutes (the transient redistribution of cPLA2 to punctate bodies isan intracellular event associated with higher activity). The activity ofcPLA₂ can also be enhanced by phosphorylation (Lin et al., 1993).Phosphorylated cPLA₂ can be distinguished from unphosphorylated cPLA₂ bymigration on SDS-PAGE. Immunoblotting revealed that cPLA₂ in controlcells was predominately unphosphorylated. Following LPS challenge cPLA₂shifted to a phosphorylated form between 10-20 minutes post-challenge.Importantly, CPLA2 inhibitors, i.e., ATFMK (arachidonyltrifluoromethylketone) and BMS 229724 have shown significant dose-dependent inhibitionof TNF-alpha and nitrite release in LPS activated microglia. Theredistribution and phosphorylation of cPLA₂ as well as, the attenuationof TNF-alpha and nitrite by cPLA2 inhibitors provide several lines ofevidence for the activation of cPLA₂ in LPS treated microglia.

[0038] COX-2 inhibitors rofecoxib (VIOXX®) and celecoxib (CELEBREX®) hadno significant effect on pro-inflammatory release on activatedmicroglia. Importantly, para-REV5901((x-pentyl-4-(3-quinolinylmethyl)benzenemethanol) a 5-LOX activatingprotein inhibitor and NDGA (nordihdroguaiaretic acid) a 5-LOX inhibitor,dose dependently inhibited TNF-alpha release and nitrite to near controllevels following LPS challenge in microglia cells.

[0039] To further validate the role of 5-LOX in pro-inflammatorycytokine release transcriptional regulators of TNF-alpha and NO wereexamined. Lipoxygenases can activate NFKB mediated transcription via thegeneration of reactive oxygen intermediates (Lee et al., 1997; Bonizziet al., 1999). Both the TNFα gene and inducible nitric oxide synthase(iNOS) gene contain NF-KB binding elements in their promoter sequencesand activation of NF-KB is crucial for gene transcription (Goldfeld etal., 1990; Drouet et al., 1991; Xie et al., 1994). Hence the effects ofinhibiting NF-KB mediated transcription using two distinct inhibitorswas assessed with BAY 11-7085 an irreversible inhibitor of IKBαphosphorylation ([IC₅₀-10μM] a biochemical event associated NF-KBactivity) and NF-KB SN-50 a cell permeable peptide which inhibitstranslocation of NF-kB active complex into the nucleus (a requiredintracellular event associated with NF-KB activity; Lin et al., 1995;Pierce et al., 1997). Both BAY 11-7085 and NF-KB SN-50 inhibited LPSinduced TNFα and NO release to control levels.

[0040] To further characterize the involvement of NF-KB in microglialsignaling, the effect of LPS on the degradation of IKBα and NF-KB (p65)translocation from the cytosol to the nucleus was also determined. Itwas observed that IKBα was rapidly degraded within 20 minutes followingLPS activation and reappeared to control levels by 60 minutes.Consistent with these observations, indirect immunofluorecence with ap65 antibody indicated that in control cells p65 was primarily localizedin the cytosol, but after stimulation with LPS p65 rapidly translocatedto the nucleus. These results demonstrate that NF-KB mediatedtranscription can play a role in microglia activation.

[0041] To determine whether cPLA₂ and 5-LOX regulate TNF(X and NOrelease by influencing NF-KB activation, the effects of cPLA₂ and 5-LOXinhibitors on IKBα degradation and nuclear translocation of NF-KB wereexamined. ATFMK and para-REV5901 attenuated the degradation of IKBαfollowing LPS stimulation. ATFMK and para-REV5901 also delayed thetranslocation of NF-KB into the nucleus. These results demonstrate thatboth cPLA₂ and 5-LOX inhibitors attenuate the release of TNFα and NO bydelaying IKBα degradation and interfering with NF-KB activation.

[0042] These data collectively represent that 5-LOX (via CPLA2, AA, andNF-KB signaling) is a preferential target over COX-2 in modulating orinhibiting microglia activation. Consequently, modulating either 5-LOXalone or in conjunction with COX-2 could have direct effects inenhancing neuronal survival in acute and chronic CNS diseases includingAlzheimer's disease, brain ischemia, traumatic brain injury, Parkinson'sDisease, Multiple Sclerosis, ALS, and subarachnoid hemorrhage.

[0043] Lin LL, Wartmann M, Lin AY, Knopf JL, Seth A, Davis RJ (1993)cPLA2 is phosphorylated and activated by MAP kinase. Cell 72:269-278.

[0044] Lee S, Felts KA, Parry GC, Armacost LM, Cobb RR (1997) Inhibitionof 5-lipoxygenase blocks IL-I beta-induced vascular adhesion molecule-1gene expression in human endothelial cells. J Immunol 158:3401-3407.

[0045] Bonizzi G, Piette J, Schoonbroodt S, Greimers R, Havard L,Merville MP, Bours V (1999) Reactive oxygen intermediate-dependentNF-kappaB activation by interleukin-lbeta requires 5-lipoxygenase orNADPH oxidase activity. Mol Cell Biol 19:1950-1960.

[0046] Goldfeld AE, Doyle C, Maniatis T (1990) Human tumor necrosisfactor alpha gene regulation by virus and lipopolysaccharide. [Proc Nat]Acad Sci U S A 87:9769-9773.

[0047] Drouet C, Shakhov AN, Jongeneel CV (1991) Enhancers andtranscription factors controlling the inducibility of the tumor necrosisfactor-alpha promoter in primary macrophages. J Immunol 147:1694-1700.

[0048] Xie QW, Kashiwabara Y, Nathan C (1994) Role of transcriptionfactor NF-kappa B/Rel in induction of nitric oxide synthase. J Biol Chem269:4705-4708.

[0049] Lin YZ, Yao SY, Veach RA, Torgerson TR, Hawiger J (1995)Inhibition of nuclear translocation of transcription factor NF-kappa Bby a synthetic peptide containing a cell membrane-permeable motif andnuclear localization sequence. J Biol Chem 270:14255-14258.

[0050] Pierce JW, Schoenleber R, Jesmok G, Best J, Moore SA, Collins T,Gerritsen ME (1997) Novel inhibitors of cytokine-induced IkappaBalphaphosphorylation and endothelial cell adhesion molecule expression showanti-inflammatory effects in vivo. J Biol Chem 272:21096-21103.

[0051] Isolation of Microglia from Rat Brains:

[0052] Rat microglia were prepared from two day old rat pups. Pup brainswere removed and the meninges were gently removed. Once sufficientamount of brains were collected, brains were minced with a bluntscissors (10 times) and transferred to a 15ml conical tube with apasteur pipette and titurated 25 times. Dissociated cells were thencentrifuged at 10000RPM for 10 minutes (RT). The supernatant was removedand 2 mls of fresh media was added. The resultant cell suspension wastiturated 10 times. Following titration the cell suspension was platedin a T175 cm2 culture flasks at a density of 4 brains per flask in 25mls. MEM media was used for the experiments, supplemented with 10% FBS,100 i.u.penicillin, 100 i.u.streptomycin and L-Glutamine. Microglia wereisolated on day 14 by shaking on an orbital rotation shaker. The purityof the cultures was 98-100% as determined by immunostaining with ED-40antibody.

[0053] Rat Microglia Cell Activation and Drug Exposure

[0054] Endotoxin (LPS) at a concentration of a 100ng/ml were used foractivation of rat microglia cells. This concentration had previouslyshown to be effective in inducing TNF-alpha and Nitrite release. Allassays were performed in 48 well plates (Becton Dickinson) at ˜2 X10⁵cells or 0.5 X10⁵ per 1 ml per well in 10% MEM media. Microglia cellswere pre-incubated lhr prior to LPS challenge with either vehicle (0.1%DMSO) or test compound in DMEM containing 10%FBS (microglia) or RPMIcontaining 10%FBS (THP-1 monocytes). Supernatants from LPS activated ratmicroglia were collected at 24 hrs post-LPS challenge.

[0055] TNF-alpha ELISA

[0056] Collected supernatants were assayed for TNF-alpha using aPharmingen OPtEIA Rat (microglia).

[0057] Nitrite Assay

[0058] Nitrite assay was performed in a 96 well plate using a ModifiedGriess Reagent (Sigma). In brief, a 100ul of Modified Griess Reagent wasadded to a 100 ul of collected supernatant. Samples were read at awavelength of 540nM. All values were calculated against a NaNO2 standardcurve.

[0059] Immunofluorescence

[0060] Cells were washed once with PBS, fixed and permeabilized with icecold methanol (100%) for 5 minutes and washed 3X in PBS for 10 min. Thecover slips were blocked for 1 hour in 10% serum/PBS (serum derived fromanimal in which secondary antibody was generated), incubated for 2-3hours in primary antibody solution (1:50 dilution in 1.5% serum/PBS) andwashed 3X in PBS for 10 min. Secondary antibody linked to fluoresceinwas applied for one hour (1:100 dilution in 1.5% serum/PBS) and washed3X in PBS for 10 min. If the nucleus was stained, the cells wereincubated for 15 minutes with DAPI (1:10000) at 37° C. and washed. Thecoverslips were then mounted onto glass slides using mounting media andviewed under a fluorescence microscope.

[0061] Immunoblotting

[0062] Immunoblotting was carried out as described previously(Parvathenani et al., 2000). Briefly 25μg of protein was fractionated ona 4-20% tris-glycine gel (NOVEX, CA) and transferred to PVDF membrane(NOVEX, CA). The membrane was probed with a polyclonal antibody specificfor IKBα. To distinguish between the phosphorylated andnon-phosphorylated forms of cPLA₂, 50μg of protein was run on an 8%tris-glycine gel (Novex, CA) for 4.5 hours at 125 V, transferred andprobed with a monoclonal antibody specific for cPLA₂.

[0063] Materials

[0064] NDGA (nordihdroguaiaretic acid), para-REV5901((x-pentyl-4-(3-quinolinylmethyl)benzenemethanol), ATFMK(arachidonyltrifluoromethyl ketone) was obtained from Calbiochem (SanDiego, Calif.). Ibuprofen and LPS was purchased from Sigma (St. Louis,Mo.). BMS 229724 was synthesized at Bristol-Myers Squibb. NF-KB SN50 and(E)3-((4-t-Butylphenyl)sulfonyl)-2-propenenitrile (BAY-11-7085) wereobtained from Biomol (Plymouth Meeting, Pa.).

[0065] Figures:

[0066] The data represents mean ±S.D. of triplicate samples of anexperiment repeated at least three times. *=Statistically significant(p<0.05) in comparison to LPS (positive control).

[0067]FIG. 1A-E Legend

[0068] Microglia were treated with 100ng/ml of LPS for various periodsof time following which A-D. cPLA₂ distribution was assessed by indirectimmunofluorescence (1A) control , (1B) LPS-15 min , (IC) LPS-15 min ,(1D) LPS-60 min, (1E) whole cell lysates were prepared and run onSDS-PAGE, transferred and probed with a cPLA₂ antibody.

[0069]FIG. 2A-D Legend

[0070] 5-lipoxygnease inhibitor (NDGA, 2A) and 5-lipoxygenase activatingprotein inhibitor (para-REV5901, 2B) significantly inhibited TNF-alpharelease, however, COX-2 inhibitors Ibuprofen (2C), Vioxx (2D), andCelebrex (2D) failed to produce any reduction in TNF-alpha release inrat primary microglia cells following LPS activation.

[0071]FIG. 3A-B Legend

[0072] cPLA2 inhibitors ATFMK (3A) and BMS-229724 (3B) significantlyinhibited TNF-alpha release in rat primary microglia cells following LPSactivation.

[0073]FIG. 4A-C Legend

[0074] cPLA2 inhibitor, ATFMK (4A) and FLAP inhibitor, para-REV5901 (4B)significantly inhibited nitrite release in rat primary microglia cellsfollowing LPS activation. However, COX-2 inhibitor, Celebrex (4C) had noeffect on nitrite release.

[0075]FIG. 5A-B Legend

[0076] Effects of NF-KB inhibitors, BAY 11-7085 and SN-50 on TNFα and NOrelease in LPS treated microglia. Microglia were treated with variousconcentrations of either BAY- or SN-50 for one hour prior to theaddition of LPS. Twenty-four hours post LPS challenge the media wasassayed for TNF(X release by ELISA (5A) and nitrite release by modifiedGreiss reagent (5B).

[0077]FIG. 6A-C Legend

[0078] Effects of cPLA₂ and 5-LOX inhibitors on LPS mediated IKBαdegradation. Microglia were treated with lOOng/ml of LPS for variousperiods of time following which whole cell lysates were prepared and runon SDS-PAGE, transferred and probed with a IKB(X antibody as mentionedin immunoblotting. (6A) 100ng/ml LPS alone, (6B) LPS +10μM ATFMK, (6C)LPS +50 μM L-655,238.

[0079]FIG. 7 A-D Legend

[0080] Effects of cPLA₂ and 5-LOX inhibitors on LPS mediated NF-KBtranslocation. Microglia were treated with 100ng/ml of LPS for variousperiods of time following which p65 distribution was assessed byindirect immunofluorescence (7A) control, (7B) LPS-5 min., (7C) LPS+10μM ATFMK-5 min., (7D) LPS +50μM L-655,238 -5min. (7E) LPS +NDGA-201M-5min.

What is claimed is:
 1. A method of modulating microglia activationcomprising the administration to a human in need thereof an effectiveamount of Zileuton®.
 2. A method of inhibiting the release ofpro-inflammatory substances from activated microglial cells comprisingthe administration to a human in need thereof an effective amount ofZileuton®.
 3. A method of treating a disorder selected from the groupconsisting of Alzheimer's disease, traumatic brain injury, brainischemia and subarachanoid hemmorhage comprising the administration to ahuman in need thereof an effective amount of Zileuton®.
 4. A method ofattenuating degradation of IkBα comprising the administration to a humanin need thereof an effective amount of Zileuton®.
 5. A method ofinhibiting nuclear translocation of the NF-kB active complex comprisingthe administration to a human in need thereof an effective amount ofZileuton®.